Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54306—Solid-phase reaction mechanisms
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/825—Pretreatment for removal of interfering factors from sample

Definitions

• This invention relates to a method for the direct determination of the free form fraction of organic substances which are present in biological fluids both in a free form and in a form bound to one or more endogenous ligands, the two forms being in equilibrium.
• endogenous ligands include without limitation proteins commonly present in said fluids.
• examples of the foregoing organic substances, which shall hereinafter be generally called analytes, are various hormones, including but not limited to T3 and T4.
• Some physiologically active substances which are present in biological fluids are balanced between a free form and a form bound to endogenous ligands present in the same fluids.
• the free-portion level is generally known to be clinically more significant than the total-substance level. Only the free form is in fact responsible for biological activity. Observations made on both normal and pathologically afflicted humans confirmed that the variations observed in free-form concentrations more closely correlate to the patients' clinical condition than are those of the entire substance. This is particularly true of thyroid and steroid hormones (Robbins J. and Rall J. E., Recent Progr. Hormone Res., 13:161 (1957); Robbins J. and Rall J. E., Physiol. Rev., 40:415 (1960)).
• T4 thyroxine
• T4 thyroxine
• T3 triiodothyronine
• this objective can be achieved by introducing a new exogenous component and having it take part in the existing equilibrium between the free analyte form and the bound analyte form, which component should have the following characteristics:
• Said method calls for a sample to be subjected to a chromatographic adsorption process using Sephadex LH-20 columns. As soon as the free analyte present in the sample is adsorbed upon the resin, its concentration in the sample is promptly restored by dissociation of the analyte from the bound portion. This adsorption and dissociation process continues until a balanced state between adsorbed analyte and free analyte in solution is reached. The quantity of adsorbed analyte is proportionate to the free-analyte concentration, and it is therefore possible to find the latter by determining the adsorbed quantity by radioimmunoassay and dividing it by the proportionality constant. This method is difficult to perform for the same reasons as all "two-step" methods (see below).
• a variation which is described in Belgian Patent BE 878687, uses an analyte-specific antibody as a sequestering agent in place of a resin.
• said antibody is introduced into a fluid, in a quantity incapable of substantially upsetting the free-analyte/bound-analyte equilibrium present in the sample, the number of antibody-binding sites which are occupied by the analyte is proportionate to the free-analyte concentration present. The number of free sites is measured after removing endogenous carrier proteins by washing and then adding a labelled analyte which enables the free-analyte concentration to be determined.
• the main drawback of the Belgian patent technique is the necessity to use an antibody with a very high affinity constant for the analyte.
• the sensitivity of this type of assay does not change in relation to the quantity of antibody used, but depends exclusively on its affinity constant. For instance:to obtain a satisfactory sensitivity when measuring free T4 an antibody with an affinity constant higher than 5 ⁇ 10 10 L/mol is required. On the other hand, if one wants to determine with acceptable sensitivity the concentration of free T3, which is present in blood in a lower concentration than T4, antibodies with an even higher affinity constant will be required.
• the European Patent Application EP 26103 describes a method for assay of free analytes which tries to avoid this two-step sequential incubation by adding an appropriately labelled analyte derivative called "analogue" to the test sample at the same time as the analyte sequestering agent (analyte-specific antibody). If a particular analogue can be selected which is capable of binding to the sequestering agent but incapable of binding to the natural ligands present in biological fluids and is, therefore, only capable of competing with the available free-analyte moiety in binding to the antibody, the level of the antibody-bound signal will change only in proportion to the free-analyte concentration in the biological fluid.
• the specific ligand is labelled, instead of the analyte derivative.
• the analyte and its derivative compete for binding to the labelled specific ligand and the measured level of specific ligand bound to the analyte derivative is used to determine the free analyte concentration.
• European Patent Application EP 155104 describes a method for free-analyte measurement in biological fluids, which uses, in addition to an analyte analogue, differential blocking agents which are intended to prevent or dissociate the bond between the analogue and natural ligands, in particular albumin.
• differential blocking agents which are intended to prevent or dissociate the bond between the analogue and natural ligands, in particular albumin.
• the addition of said differential blocking agents to the sample to be measured substantially affects the free-analyte/bound-analyte equilibrium in the sample itself, which distorts the measured levels.
• An object of the present invention is, therefore, to provide a method for the determination of the free portion of analytes present in biological fluids, which provides a solution to at least one of the above-mentioned problems.
• Another object is to provide a method that has the reliability of known "two step” methods, it has adequate sensitivity and is easy to perform using routine laboratory procedures.
• the method of the invention can be used to measure the free-form concentration not only of analytes such as thyroid hormones (thyroxine and triiodothyronine) and steroid hormones (e.g. aldosterone, testosterone, cortisol), but also of all the substances which are present in biological fluids and have a bound form and a free form balanced with each other (e.g.: biochemical messengers, drugs and their metabolites, polypeptides and proteins, vitamins, polysaccharides, tumor antigens, toxins, alkaloids and the like).
• analytes such as thyroid hormones (thyroxine and triiodothyronine) and steroid hormones (e.g. aldosterone, testosterone, cortisol), but also of all the substances which are present in biological fluids and have a bound form and a free form balanced with each other (e.g.: biochemical messengers, drugs and their metabolites, polypeptides and proteins, vitamins, polysacc
• the present method for determining the amount of an analyte (A) present in a biological fluid in free form comprises the following elements:
• a first exogenous ligand (L1) capable of sequestering an analyte quantity correlated to the free portion present in a biological fluid
• a dissociating agent (D) capable of dissociasing the sequestered analyte from the first exogenous ligand
• SS standard solutions
• the method is implemented by a "competitive" assay system where both exogenous ligands L1 and L2 are simultaneously present and easy to separate from the other reaction components.
• a first exogenous ligand (L1) capable of sequestering an analyte quantity proportionate to the free-analyte concentration present in a biological fluid and then to release said quantity pursuant to the action of the dissociating agent (D)
• L1 a first exogenous ligand capable of sequestering an analyte quantity proportionate to the free-analyte concentration present in a biological fluid and then to release said quantity pursuant to the action of the dissociating agent (D)
• the quantity of the second specific ligand may, therefore, be kept lower than the quantities of the labelled analyte and of the sequestered analyte in the prior art methods. This results in higher sensitivity of the present method compared to the common "two-step" assay methods which use the same specific antibody both in the sequestering and measuring steps.
• a dissociating agent (D) capable of releasing the sequestered analyte and not interfering with the competition reaction between dissociated analyte and labelled analyte in binding to the second exogenous ligand, allows both ligands to be simultaneously present during all the measuring stages, with consequent simplification of the operating procedure as compared to the prior art "two step" methods which use different systems or techniques in performing the sequestering step from those used in performing the measuring step.
• the method of the present invention comprises the use of the following components:
• L1--a first specific exogenous ligand preferably bound to the surface of a stationary or dispersed solid phase
• L2--a second specific exogenous ligand preferably bound to the surface of a stationary or dispersed solid phase
• D--an agent capable of dissociating the analyte A from the first exogenous ligand L1, without affecting the ability of the second exogenous ligand L2 to bind to said analyte and to the labelled analyte M;
• SS--a set of standards comprising, e.g., solutions or sera with known free-analyte concentrations or a set of standard concentration values (e.g., in table or graph form).
• ligands L1 and L2 are present in quantities incapable of changing to any significant extent (e.g., keep the change to no more than 10%) the concentration of the free analyte A present in the biological fluid and, in particular, the second ligand L2 is deficient compared to the quantity of the labelled analyte M (e.g., no more than enough L2 to bind about 50-60% of M).
• the percentage of the sequestered analyte compared with total analyte must be evaluated. This percentage roughly correlates with the percentage decrease of the free analyte concentration due to the presence of the "new" ligands L1 and L2 in the system (See, Ligand Quarterly, 8 (Suppl. to No. 1), 1989).
• the first exogenous ligand L1 is selected among those capable of sequestering the analyte A without substantial interference from any other substances which might compete with the analyte binding to L1. (This means that L1 binds A quantitatively with relatively high affinity and with specificity.) The amount of the sequestered analyte A is thus proportionate only to the concentration of the free analyte A.
• the analyte A present in the biological fluid also binds to the second exogenous ligand L2, but to a lower extent quantitatively before dissociation takes place.
• L1 and L2 are chosen so that e.g., only a certain amount of analyte binds to L2 and a multiple of that amount (e.g., 2-5 times) is bound to L1.
• This can be ascertained beforehand using, e.g., labelled analyte M and can be accomplished either by choice of relative affinities of L1 and L2 for A or by choice of the relative quantities of these exogenous ligands, or both.
• L2 has to have sufficient affinity for A to produce a differential "reading" in the assay and for the same reason must be present in a sufficiently high quantity.
• sequestered quantity of the analyte initially bound to L2 is also proportionate to the concentration of the free analyte and therefore easily standardized.
• the dissociating agent (D), which dissociates the analyte A from the first ligand L1 may be either a substance capable of successfully competing with the analyte A in binding to the same site(s) of the first exogenous ligand L1, but not to those of the second exogenous ligand L2, or an agent capable, because of its physico-chemical characteristics, to weaken considerably the bond between the analyte A and the first ligand L1 without, however, substantially affecting the affinity characteristics of the second specific ligand L2 for the analyte.
• the binding affinity of L1 for A should be at least 100 times lower than the binding affinity of L1 for A, when D is not present.
• Relative affinity characteristics can be worked out as a function of the equilibria and substances involved using commercially available software such as EUREKA, available from Real Software, Sequel, Calif.
• a solid phase as a support for the exogenous ligands allows, after the sequestering step, a rapid and easy removal of the natural ligands present in the sample and a simple and rapid separation, during the measuring step, of the labelled analyte bound to the second exogenous ligand from the still-free labelled analyte.
• Precipitation of L2 complexes is another of the possible alternatives for separation from the natural ligands but use of a solid phase support is preferred.
• the solid phase may be stationary, e.g., the walls of the very container in which the determination takes place, such as for instance: test tubes of polystyrene, polypropylene and any other material capable of causing adhesion of exogenous ligands.
• the solid phase may be suspended, e.g., consist of appropriate elements which serve to increase the usable surface area (such as beads). These elements are introduced into the container and can be made of the same materials as illustrated above. These materials are commercially available, e.g., from Pierce Chemical Co., Richford, Ill. and Precision Plastic Ball Co., Chicago, Ill. Another type of support that can be used is, e.g. that used in ELSA (TM) Solid Phase from Int'l CIS, Gif-Sur-Yvette, France.
• TM ELSA
• the solid phase may also be dispersed, as is the case, for instance, when using cellulose microelements or the like which may then be separated by centrifugation, by the action of a magnetic field or by any other technique which makes use of their typical physico-chemical properties, such as chromatography columns.
• the advantage of a stationary solid phase is that it can be more easily separated from the reaction mixture during determination.
• the advantage of a dispersed solid phase is that it has a larger surface available to fix exogenous ligands. Choice between them is within the skill in the art.
• the exogenous ligands may be selected either among natural binding proteins, such as (in the case of thyroxin measurements) TBG (thyroxin binding globulin), transcortin, specific receptors and the like, or among analogues thereof obtained e.g. by DNA recombinant techniques and the like.
• Polyclonal as well as monoclonal analyte-specific antibodies can also be exogenous ligands.
• Analyte specific antibodies can easily be produced by well-known methods. Nonlimiting examples of ligands can be found, e.g., in Weeks; Avrameas; Ishikawa; Hunter, all of them infra.
• Fixation of ligands L1 and L2 to the solid phase may be direct or indirect.
• Indirect fixation involves binding to the solid phase by known techniques a molecular species which has an affinity for a specific tracer (label) which in turn is fixed to a ligand.
• the sequence (solid phase)-(streptavidin)/(biotin)-(ligand) or (solid phase)-(antifluorescein antibody)/(fluorescein)-(ligand) can be used.
• the ligand (L1 and/or L2) is an antibody
• it can also be fixed to the solid phase by the use of anti-antibody antibodies, e.g.: (solid phase)-(anti-rabbit gamma globulin)/(anti-analyte antibody produced in rabbits).
• the two exogenous ligands used may be bound to one or more different solid phases using either the same method or different methods.
• labelled analyte M one may resort to the usual techniques well-known to those skilled in the art and described in the literature, which include: labelling with a radioactive atom (for instance 125 I), labelling with enzymes or components of enzyme systems and labelling with chemiluminescent or fluorescent groups such as for instance acridinium or fluorescein esters. See, e.g., Weeks, I. et al. Clin. Endocrinol. 20:489, 1984; Avrameas, S. Immunochem. 6:43, 1969; Ishikawa, E. Develop. Immunol. 18:219, 1983; Hunter, W. M. Nature 194:495, 1962. In general, any material that can bind firmly to the analyte and that can be detected can be used as a label.
• a radioactive atom for instance 125 I
• enzymes or components of enzyme systems labelling with chemiluminescent or fluorescent groups such as for instance acridinium or fluorescein
• Substances that function as selective dissociating agents may be found among synthetic or natural compounds with structures capable of giving them a binding affinity only for the first exogenous ligand.
• this substance may be selected in a group including 8-anilino-1-naphthalenesulphonic acid (ANSA), sodium salicylate, sodium ethyl-[2-mercaptobenzoate(2-)-O,S]-mercurate(1-) (Thimerosal) and the like.
• ANSA 8-anilino-1-naphthalenesulphonic acid
• sodium salicylate sodium ethyl-[2-mercaptobenzoate(2-)-O,S]-mercurate(1-) (Thimerosal) and the like.
• dissociating function when the dissociating function is based on physico-chemical characteristics, such characteristics may include:pH, ionic strength, temperature and generally any variable capable of loosening binding forces.
• characteristics may include:pH, ionic strength, temperature and generally any variable capable of loosening binding forces.
• An example of the use of such agent in the form of a solution is described in Example 2.
• the above mentioned dissociating agent and labelled analyte may both be present in the same solution, in order to further simplify the measuring process.
• T4 free thyroxine
• T3 free triiodothyronine
• T3 and T4 are known to circulate in blood mainly in a protein-bound form, but a free portion of these hormones is also known to be present in blood and is found to be in thermodynamic equilibrium with the bound portion.
• this hormone is bound to the extent of about 70% to thyroxine binding globulin (TBG), about to thyroxine binding prealbumin (TBPA) and about 10% to albumin.
• the free T4 in blood is approximately 0.02-0.03% of the total hormone concentration.
• T3 is carried by the carrier proteins themselves (TBG:60%, TBPA:15%, albumin:25%), while the free form accounts for about 0.3% of the total hormone concentration.
• TSG human thyroxine binding globulin
• ANSA magnesium salt of 8-anilino-1-napththalenesulphonic acid
• gelatin for microbiology HEPES (4-(2-hydroxyethyl)-1-piperazinyl) ethanesulphonic acid), HEPES sodium salt, thyroxine (T4), 3,3', 5-triiodothyronine (T3) (SIGMA, St Louis, Missouri).
• the solid phase which consisted of 12 ⁇ 75 mm polystyrene test tubes with both the exogenous ligand L1 (TBG) and the exogenous ligand L2 (anti-thyroxine antibody produced in rabbits) adhering to their inner surfaces, was prepared using the following operating procedure:
• each test tube is washed with 1 mL of 0.05M phosphate buffer at pH 7.5 and 0.1% sodium azide.
• each test tube is washed with 1 ml of 0.05M phosphate buffer at pH 7.5 containing 1% gelatin and 0.1% sodium azide.
• the rabbit anti-rabbit gamma globulin antibody and the anti-T4 antibody were prepared using methods well known to those skilled in the art and described in the literature. Vaitukaitis, G. G. Clin. Endocr. 33:988, 1971; Chapman, R. S. in Hunter, W. M. "Immunoassays for Clinical Chemistry", p. 456, 1983, Churchill et al. Eds, Edinburgh (U.K.); Marguerita, S. Experientia 37:314, 1981. The two antisera produced were then purified, the former by affinity chromatography, the latter by specific gamma-globulin precipitation with ammonium sulphate. Such antibodies are also commercially available, e.g. from Boehringer Mannheim, Mannheim, Germany; or Scantibodies Lab. Inc., Santee, Calif.
• the thyroxine binding globulin (TBG) was treated with biotin using biotin-hydroxysuccinimide ester and the extent of the biotin treatment was determined after ascertaining the relationship existing between the biotin/TBG ratio and the ability of the biotinylated binding protein to bind the analyte.
• the ability of the biotinylated TBG to bind the analyte was evaluated by adding to the test tube, previously sensitized with constant quantities of TBG-biotin with different biotin/TBG ratios, 1 mL of a T4- 125 I solution (100 pmol/L-specific activity: 2,000 ⁇ Ci/ ⁇ g) in 0.2M HEPES buffer at pH 7.4. After incubation for one hour at room temperature, shaking at 100-150 rpm, the radioactivity not bound to TBG was removed by suction and washing and the bound radioactivity was measured by a gamma-counter. The highest binding ability was obtained using a biotin/TBG ratio of about 3. (It is evident that once such a ratio has been optimized this step need not be performed.)
• the labelled analyte (T4- 125 I) was prepared using well known procedures and diluted in a dissociating solution made up of 0.05M TRIS buffer at pH 8.6 containing 0.02% ANSA, 0.1% gelatin and 0.1% sodium azide.
• Standard (reference) solutions were prepared adding to a T4-free human serum (obtained from normal human serum, deprived of T4 by treatment with carbon-dextrane) respectively 15, 30, 50, 80, 120 ng/mL of T4.
• Each of 200 ⁇ L of standard solution and 200 ⁇ L of a serum to be determined containing the analyte (A) are added respectively to a coated tube sensitized as described in Part 1 (i.e., having L1 and L2 adhering to their surface).
• test tubes After incubation for 1 hour at room temperature shaking at 100-150 rpm, the solution is thoroughly removed by suction or decantation and the test tubes are washed twice with 0.05M phosphate buffer at pH 7.5 containing 0.1% sodium azide.
• the standard curve is calculated and the levels relative to the samples containing the serum to be determined are obtained by curve fitting.
• Table I shows an example of standard curve.
• the radioactive solution is decanted and the test tubes are washed twice with 1 mL of 0.05M phosphate buffer at pH 7.5 containing 0.1% sodium azide.
• a dissociating solution (D) consisting of 0.05M TRIS buffer at pH 8.6 containing 0.02% ANSA is added to each test tube.
• test tubes After incubation for 15 minutes at room temperature, the added solution is removed by suction and, after washing with 1 mL of 0.05M phosphate buffer at pH 7.5 containing 0.1% sodium azide, the test tubes are again measured by a gamma-counter.
• the last radioactivity measured correlates to the quantity of non-dissociated radioactive analyte.
• Table IV a) shows the results obtained in the dilution test which was carried out by adding to the solid phase 200 ⁇ L of sample at different known dilutions.
• Table IV b) shows the results obtained by adding to the solid phase different known volumes of the same sample.
• Table V shows the results relative to a standard curve obtained using the same specific antibody L2 in both the sequestering and measuring steps (Method A), and compares them with those obtained by the method which is an object of the present invention (Method B), which uses two different ligand systems, one for sequestration (TBG) and the other for measurement (antibody) L2.
• the solid phase which consisted of 12 ⁇ 75 mm polystyrene test tubes with the two exogenous ligands (L1 and L2) adhering to their inner surfaces was prepared using the following operating procedure:
• each test tube is washed with 1 mL of 0.05M phosphate buffer at pH 7.5 containing 0.1% sodium azide.
• the monoclonal anti-T3 antibody and the polyclonal anti-T3 antibody bind respectively to the anti-mouse gamma globulin antibody and to the anti-rabbit gamma globulin antibody previously bound to the test tube inner walls.
• the obtained solution is removed by suction and the test tubes are washed with 1 mL of 0.05M phosphate buffer at pH 7.5 containing 1% gelatin and 0.1% sodium azide.
• Table VI shows the results related to T3- 125 I binding, under different pH conditions, to the two antibodies used.
• the standard solutions were prepared adding scalar quantities respectively of 0.15, 0.36, 0.77, 1.05, 3.75 ng/mL of T3 to a T3-free human serum obtained from normal human serum previously deprived of T3 by treatment with carbon-dextrane.
• the free-T3 concentration present in the solutions was determined using the reference method described in the U.S. Pat. No. 4,255,574.
• Standard sera or unknown sera (500 ⁇ L each) are added respectively to a coated tube.
• reaction mixture After incubation for 1 hour at room temperature shaking at 100-150 rpm, the reaction mixture is removed by suction or decantation and the test tubes are washed twice with 0.05M phosphate buffer at pH 7.5 containing 0.1% sodium azide.
• a dissociating solution made up of 0.1M citrate buffer at pH 3.5, 0.1% gelatin and 0.1% sodium azide, containing 8 pmol/L of labelled analyte (about 40,000 cpm-specific activity: 3,352 ⁇ Ci/ ⁇ g) is added to each test tube.
• reaction mixture After incubation for 1 hour at room temperature shaking at 100-150 rpm, the reaction mixture is removed by suction or decantation and the radioactivity bound to the test tubes is measured by a gamma-counter.
• the standard curve is calculated and the levels of unknown sera are obtained by curve fitting.
• Table VII shows an example of standard curve.
• Serum specimens can be tested without pre-treatment. They should be stored at 2°-8° C. for up to 24 hours prior to testing, or at -20° C. for up to 5 months. Testing should be at room temperature.
• the assay reagents are:
• Labelled Analyte and Dissociating Agent 125 I-T-4 (105 mL; L1) in 0.05 m Tris-EDTA buffer, pH 8.6, and also containing 0.02% ANSA (D), BSA, 0.1% sodium azide and red coloring.
• L1/L2:50 polystyrene tubes (12 ⁇ 75 mm) are provided. They are coated with anti-T4 polyclonal antibody (L2) and TBG (L1) (same amounts as in Example 1). Uncoated tubes are also used to assay for 125 I-T4 total activity.
• Free T4 Standards for Calibration These are supplied in labelled vials in freeze-dried form and include a "standard zero" and five standards containing 0.1% sodium azide and known amounts of T4 in serum, as follows:
• the T4 standards should be reconstituted in 2.0 mL distilled water before use.
• Control Human Sera These are pools of untreated human sera supplied in freeze-dried form and should be reconstituted in 1.0 mL distilled water. They contain precalibrated free T4 and 0.1% sodium azide and are used to test accuracy of the present assay.
• Washing Buffer 100 mL (to be diluted 1:5 with de-ionized or distilled water) containing 0.25M phosphate buffer, pH 7.5, and 0.1% sodium azide.
• All reagents should be stored at 2°-8° C. After reconstitution, the free T4 standard can be stored at 2°-8° C. for up to 14 days and the washing buffer for up to 30 days.
• the control sera should be treated as the serum samples.
• All reagents and samples should be brought to room temperature.
• the working solution is made up and the other freeze-dried reagents are reconstituted by adding the prescribed amount of water, waiting 20 minutes at room temperature and mixing gently. All tests are performed in duplicate.
• Liquid is removed thoroughly from all tubes by aspiration and the tubes are rinsed twice with 1 mL of diluted washing buffer. Liquid is aspirated after each wash. Labelled analyte preparation (1 mL) is added to each tube and the tubes are mixed and incubated as above. In this step, the dissociating agent will displace free T4 from L1 and it will compete with labelled T4 for binding to L2. Unbound ligand is thoroughly removed by aspiration. The bound radioactivity in each tube is measured ( 125 I).

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